Protochlamydia Naegleriophila Antigen Was Tested with Serum Against All These Different Sp

DISPATCHES

(7). Ameba fi lled with bacteria exhibiting 3 developmental Protochlamydia stages already described in other Parachlamydiaceae (8) were observed (Figure 1). naegleriophila To measure the serologic differentiation index (SDI) between strain KNic and other Chlamydia-like organisms, as Etiologic Agent we immunized Balb/c mice to produce anti-KNic antibodies. Purifi ed Pr. amoebophila (ATCC PRA-7), Simkania of Pneumonia negevensis (ATCC VR-1471), Parachlamydia acanth- Nicola Casson,* Rolf Michel,† Karl-Dieter Müller,‡ amoebae strain Seine, Waddlia chondrophila (ATCC 1470), John David Aubert,* and Gilbert Greub* Neochlamydia hartmannellae (ATCC 50802), Criblamydia sequanensis (CRIB 18), and Rhabdochlamydia crassifi cans Using ameba coculture, we grew a Naegleria endo- (CRIB 01) antigens were tested by micro-immunofl uores- symbiont. Phenotypic, genetic, and phylogenetic analyses cence against mouse anti-KNic antibodies, whereas KNic supported its affi liation as Protochlamydia naegleriophila antigen was tested with serum against all these different sp. nov. We then developed a specifi c diagnostic PCR for Protochlamydia spp. When applied to bronchoalveolar la- Chlamydia-like organisms (9). SDIs were calculated as vages, results of this PCR were positive for 1 patient with described (9,10). Serum from mice immunized with KNic pneumonia. Further studies are needed to assess the role showed strong reactivity against autologous antigen (titers of Protochlamydia spp. in pneumonia. of 4,096). Signifi cant cross-reactivity between KNic and Pr. amoebophila (SDI = 7) and P. acanthamoebae (SDI = 10) was observed. Mouse anti-KNic serum did not react ecently, a Naegleria endosymbiont (KNic) was ob- with other Chlamydia-like organisms (Table 1). Because Rserved but remained uncultivable, precluding precise cross-reactivity between members of the order Chlamydi- identifi cation (1). We grew a large amount of strain KNic ales was proportional to the relatedness between each spe- by using Acanthamoeba castellanii, which enabled phe- cies (9), the strong cross-reactivity between KNic and Pr. notypic, genetic and phylogenetic analyses that supported amoebophila supports the affi liation of KNic in the genus its affi liation as Protochlamydia naegleriophila. This new Protochlamydia. ameba-resistant intracellular bacteria might represent a Taxonomic position of KNic was further defi ned by new etiologic agent of pneumonia because it is likely also sequencing 16Sr RNA (rrs, DQ635609) and ADP/ATP resistant to human alveolar macrophages (2,3). Because translocase (nnt, EU056171) encoding genes. The rrs other Parachlamydiaceae were associated with lung infec- was amplifi ed/sequenced using 16SIGF/RP2Chlam prim- tion (4–6), we assessed the role of Pr. naegleriophila in ers (11). The nnt was amplifi ed/sequenced using nntF2p pneumonia by developing a diagnostic PCR and applying (5′-TGT(AT)GAT(CG)CATGGCAA(AG)TTTC-3′) and it to bronchoalveolar lavages. nntR1p (5′-GATTT(AG)CTCAT(AG)AT(AG)TTTTG-3′) primers. Genetic and phylogenetic analyses were conducted The Study by using MEGA software (12). The 1,467-bp rrs sequence KNic growth in A. castellanii was assessed by immu- showed 97.6% similarity with Pr. amoebophila, 91.8%– nofl uorescence (7) with in-house mouse anti-KNic and Al- 93.2% with other Parachlamydiaceae, and 85.7%–88.6% exa488-coupled anti-immunoglobulin antibodies (Invitro- with other Chlamydiales. Based on the Everett genetic gen, Eugene, OR, USA). Confocal microscopy (LSM510; criteria (13), KNic corresponds to a new species within Zeiss, Feldbach, Switzerland) confi rmed the intracellular the Protochlamydia genus because its sequence similarity location of KNic and demonstrated its rapid growth within with Pr. amoebophila is >95% (same genus) and <98.5% A. castellanii. To precisely assess the growth rate, we per- (different species). Phylogenetic analyses of rrs gene se- formed PCR on A. castellanii/KNic coculture by using PrF/ quences showed that KNic clustered with Pr. amoebophila, PrR primers and PrS probe. After 60 hours, we observed with bootstraps of 98% and 95% in neighbor-joining and an increased number of bacteria per microliter of 4 loga- minimum-evolution trees, respectively. The 569-bp nnt se- rithms (online Appendix Figure, available from www.cdc. quence exhibited 91.1% similarity with Pr. amoebophila, gov/EID/content/14/1/167-appG.htm). 65.5%–72.6% with other Parachlamydiaceae, and 55.4%– A. castellanii/KNic and N. lovaniensis/KNic cocul- 72.6% with other Chlamydiales. Phylogenetic analyses of tures were processed for electron microscopy as described nnt sequences showed that KNic clustered with Pr. amoe- *University Hospital Center and University of Lausanne, Lausanne, bophila. On the basis of these analyses, we propose to name Switzerland; †Central Institute of the Federal Armed Forces Medi- strain KNic “Protochlamydia naegleriophila.” cal Services, Koblenz, Germany; and ‡Institut für Medizinische Mi- We then developed a specifi c diagnostic PCR for krobiologie der Universität Essen, Essen, Germany Protochlamydia spp. Primers PrF (5′-CGGTAATACG

168 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 1, January 2008 Protochlamydia naegleriophilIa and Pneumonia

Figure 1. Transmission electron microscopy of Protochlamydia naegleriophila. A) Naegleria lovaniensis trophozoite after transfer of endocytobionts; strain KNic (p) from the original host strain showing 15 coccoid bacteria distributed randomly within the cytoplasm of the host ameba. N, nucleus; en, endosome (karyosome) within the nucleus; v, empty food vacuoles. Magnifi cation ×10,500; bar = 1 μm. B) N. lovaniensis trophozoite jammed with numerous endoparasitic stages of Pr. naegleriophila. Magnifi cation ×16,800; bar = 1 μm. C) Enlarged detail of N. lovaniensis trophozoite with intracytoplasmic stages of Pr. naegleriophila. Some stages show binary fi ssion indicated by the fi ssion furrow (arrows). The endoparasites have a wrinkled gram-negative outer membrane rendering a spiny appearance to the endoparasites. Signs of damage are obvious within the cytoplasm of the host ameba. mi, mitochondria. Magnifi cation ×43,500; bar = 0.5 μm. D) Pr. naegleriophila within vacuoles of Acanthamoebae castellanii ameba 2 days postinfection. Elementary bodies (eb) and reticulate bodies (rb) are visible. Elementary bodies harbor a smooth membrane compared with the reticulate bodies, which have a spiny shape. Magnifi cation ×10,000; bar = 2 μm. E) Enlarged detail of A. castellanii trophozoite with intracytoplasmic stages of Pr. naegleriophila 3 days postinfection. Binary fi ssion is observed (di). Magnifi cation ×20,000; bar = 1 μm. F) Crescent body (arrow) within A. castellanii observed 3 days postinfection. Magnifi cation ×20,000; bar = 1 μm.

GAGGGTGCAAG-3′) and PrR (5′-TGTTCCGAGGTT a Bland-Altman bias of 0.99 and a limit of agreement of GAGCCTC-3′) as well as probe PrS (5′-TCTGACTGAC 2.87 (Figure 2, panel A). Inter-run variability was low at ACCCCCGCCTACG-3′) were selected. The 5′-Yakima- high concentration, 1.12, 1.71, 0.82, 1.77 cycles for 105, Yellow probe (Eurogentec, Seraing, Belgium) contained 104, 103, 102 copies/μL, respectively. Inter-run variability locked nucleic acids (underlined in sequence above). The was higher at low concentration, 4.22 cycles for 101 cop- reactions were performed with 0.2 μM each primer, 0.1 ies/μL (Figure 2, panel A). Analytical specifi city was tested μM probe, and iTaqSupermix (Bio-Rad, Rheinach, Swit- with bacterial and eukaryotic DNA (Table 2). The PCR zerland). Cycling conditions were as described (14), and slightly amplifi ed DNA from R. crassifi cans, another Chla- PCR products were detected with ABIPrism7000 (Applied mydia-like organism. No cross-amplifi cation was observed Biosystems, Rotkreuz, Switzerland). Each sample was am- with any other bacteria or with human cells. The absence plifi ed in duplicate. Inhibition, negative PCR mixture, and of cross-amplifi cation of P. acanthamoebae is important extraction controls were systematically tested. because this Chlamydia-related bacteria is considered an To allow quantifi cation, a plasmid containing the tar- emerging agent of pneumonia (4–6). get gene was constructed by cloning PCR products into We tested 134 bronchoalveolar lavage samples from pCR2.1-TOPO vector (Invitrogen, Basel, Switzerland). patients with (n = 65) and without (n = 69) pneumonia and Recombinant plasmid DNA quantifi ed using Nanodrop extracted DNA by using a Bio-Rad Tissue Kit. One sample ND-1000 (Witech, Littau, Switzerland) was 10-fold diluted was positive, with 543 and 480 copies/μL. This positive and used as positive controls. result was confi rmed using the 16sigF/16sigR PCR (13), The analytical sensitivity was 10 copy/μL (Figure 2, which targets another DNA segment. This sequence ex- panel A). Intra-run variability was good (Figure 2B) with hibited 99.6% (284/285) similarity with Pr. naegleriophila

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Table 1. Antibody titers and serologic differentiation index (SDI) obtained from reciprocal cross-reactions of mouse antiserum with different Chlamydia-like organisms, as determined by immunofluorescence* Antigen titers (SDI) P.a. Strains Pr. Pr. Simkania strain Waddlia Neoclamydia Criblamydia R. naegleriophila amoebophila negevensis Seine chondrophila hartmannellae sequanensis crassificans Pr. 4,096 (0) 256 (7) <4 (14) 512 (10) 4 (20) 16 (14) <4 (20) <4 (13) naegleriophila Pr. 32 (7) 256 (0) <32 (5) 1,024 64 (10) 32 (0) 128 (9) 64 (3) amoebophila (3) S. negevensis 32 (14) 128 (5) 512 (0) 64 (12) 128 (12) <32 (12) 512 (8) 256 (3) P. strain 128 (10) 512 (3) 32 (12) 16,384 64 (19) 64 (12) <32 (19) <32 (12) Seine (0) W. 32 (20) 128 (10) 32 (12) <32 (19) 32,768 (0) <32 (18) 512 (13) 128 (10) chondrophila N. 32 (14) 128 (0) <32 (12) 128 (12) <32 (18) 2,048 (0) <32 (18) <32 (11) hartmannellae C. 32 (20) 128 (9) 128 (8) 64 (19) 256 (13) <32 (18) 32,768 (0) 128 (8) sequanensis R. 32 (13) 128 (3) 64 (3) 64 (12) 64 (10) <32 (11) 256 (8) 256 (0) crassificans *Pr., Protochlamydia; P.a., Parachlamydia acanthamoebae; R., Rhabdochlamydia. Titers highlighted in gray, previously published in (9), are provided here because these data were used to calculate SDI between Pr. naegleriophila strain KNIC and the other Chlamydia-related bacteria. strain KNic and 95.1% (269/283) with Pr. amoebophila. agent was identifi ed despite extensive microbiologic inves- The presence of Protochlamydia antigen in the sample was tigations of bronchial aspirate and bronchoalveolar lavage. confi rmed by immunofl uorescence performed using rabbit Results of Gram stain, auramine stain (for Mycobacterium anti-KNic antibody directly on the bronchoalveolar lavage spp.), and silver stain (for Pneumocystis carinii) tests were sample and by ameba coculture (online Appendix Figure). negative. Only physiologic oropharyngeal fl ora could be The positive sample was taken from an immunocom- grown on sheep-blood and chocolate-bacitracin agars. promised patient who had cough, dyspnea, and a lung in- Cell culture, as well as culture for fungi and mycobacteria, fi ltrate. Bronchoscopy examination of the lower respiratory remained sterile. Moreover, results of PCRs specifi c for tract showed mucosal infl ammation localized at the middle the detection of Legionella pneumophila, Chlamydophila lung lobe. Cytology and Gram stain of the bronchoalveolar pneumoniae, and Mycoplasma pneumoniae (15) were all lavage showed many leucocytes with macrophages (65%) negative. The patient recovered and remained free of symp- and neutrophils (23%). Although no antimicrobial treatment toms of acute lung infection during the next 20 months. was administered prior to bronchoscopy, no other etiologic

Figure 2. A) Intra and inter-run reproducibility of the real-time PCR assessed on duplicate of plasmidic positive controls performed at 10-fold dilutions from 105 to 101 plasmid/μL during 6 successive runs. Standard deviations show the intra-run reproducibility of the real- time PCR. B) Plots of the cycle threshold (Ct) of fi rst and second duplicates, showing intra-run and inter-run variability of the real-time PCR between duplicates of positive control. 95% confi dence interval is shown by the dashed lines. C) Bland-Altman graph showing the difference of Ct of both duplicates according to the mean of the Ct of duplicates. The dashed line shows the 95% confi dence interval (i.e., limit of agreement).

170 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 1, January 2008 Protochlamydia naegleriophilIa and Pneumonia

Table 2. Bacterial and eukaryotic DNA used to determine the developmental cycle, with reticulate, elementary, and cres- specificity of the real-time PCR cent bodies. The reticulate body is about 900 nm and has a Bacterial DNA Source/strain spiny appearance similar to that of P. amoebophila (Figure Bordetella pertussis Clinical specimen 2, panel B). To be classifi ed within the Pr. naegleriophila Chlamydia trachomatis Clinical specimen species, a new strain should show a 16Sr RNA similarity Chlamydophila pneumoniae ATCC VR-1310 Criblamydia sequanensis CRIB-18 >98.5% (13) and similar phenotypic traits. Enterococcus faecalis ATCC 29212 Escherichia coli ATCC 35218 Acknowledgments Gardnerella vaginalis Clinical specimen We thank J.L. Barblan and the staff of Pôle Facultaire Médi- Haemophilus influenzae ATCC 49247 cal Universitaire at the Medical Faculty of Geneva for assisting Klebsiella pneumoniae ATCC 27736 with electron microscopy analysis, staff of the Cellular Imaging Lactobacillus spp. Clinical specimen Facility for assisting with confocal microscopy analysis, Gerhild Legionella pneumophila Clinical specimen Gmeiner for technical assistance in preparing Naegleria lovani- Listeria monocytogenes Clinical specimen Moraxella catharralis Clinical specimen ensis with Protochlamydia strain KNic for electron microscopy, Mycobacterium tuberculosis Clinical specimen Philip Tarr for reviewing the manuscript, and M. Perrenoud and Neisseria lactamica Clinical specimen S. Aeby for technical help. Neisseria weaveri Clinical specimen Neochlamydia hartmanella ATCC 50802 This work was supported by the Swiss National Science Parachlamydia acanthamoebae strain BN9 ATCC VR-1476 Foundation grants FN 3200BO-105885 and FN 3200BO-116445. Parachlamydia acanthamoebae strain ATCC VR-1476 G.G. is supported by the Leenards Foundation through a career Hall’s coccus award entitled “Bourse Leenards pour la relève académique en Protochlamydia amoebophila strain ATCC PRA-7 médecine clinique à Lausanne. UWE25 Pseudomonas aeruginosa ATCC 27853 Ms Casson is completing a PhD thesis at the University of Rhabdochlamydia crassificans CRIB-01 Lausanne. Her research is dedicated to defi ning the role of the Simkania negevensis ATCC VR-1471 obligate intracellular Chlamydia-like organisms to humans, dis- Staphylococcus epidermidis Clinical specimen covering new species, and defi ning their role in pneumonia. Streptococcus agalactiae ATCC 13813 Streptococcus mutans Clinical specimen Streptococcus pneumoniae Clinical specimen References Streptococcus pyogenes ATCC 19615 Waddlia chondrophila ATCC VR-1470 1. Michel R, Muller KD, Hauröder B, Zöller L. A coccoid bacterial Eukaryotic DNA Source/strain parasite of Naegleria sp. (Schizopyrenida: Vahlkampfi idae) inhib- Acanthamoeba castellanii ATCC 30010 its cyst formation of its host but not transformation to the fl agellate Candida albicans ATCC 10231 stage. Acta Protozool. 2000;39:199–207. Human cells ATCC CCL-185 2. Greub G, Mege JL, Raoult D. Parachlamydia acanthamebae enters and multiplies within human macrophages and induces their apopto- Conclusions sis. Infect Immun. 2003;71:5979–85. Isolating new species from environmental and clini- 3. Greub G, Raoult D. Microorganisms resistant to free-living amoebae. Clin Microbiol Rev. 2004;17:413–33. cal samples is important to better defi ne their epidemiol- 4. Greub G, Raoult D. Parachlamydiaceae: potential emerging patho- ogy and potential pathogenicity. We defi ned the taxonomic gens. Emerg Infect Dis. 2002;8:625–30. position of a novel Naegleria endosymbiont and proposed 5. Greub G, Berger P, Papazian L, Raoult D. Parachlamydiaceae as its affi liation within the Protochlamydia genus as Pr. nae- rare agents of pneumonia. Emerg Infect Dis. 2003;9:755–6. 6. Greub G, Boyadjiev I, La Scola B, Raoult D, Martin C. Serologi- gleriophila sp. nov. Moreover, we developed a new PCR cal hint suggesting that Parachlamydiaceae are agents of pneumo- targeting Protochlamydia spp., applied it to clinical sam- nia in polytraumatized intensive care patients. Ann N Y Acad Sci. ples, and identifi ed a possible role of Pr. naegleriophila as 2003;990:311–9. an agent of pneumonia. 7. Casson N, Medico N, Bille J, Greub G. Parachlamydia acanthamoebae enters and multiplies within pneumocytes and lung fi broblasts. Protochlamydia naegleriophila (nae.gle.rio′.phi.la Microbes Infect. 2006;8:1294–300. Gr. fem.n. Naegleria, name of host cell, Gr. adj. philos, 8. Greub G, Raoult D. Crescent bodies of Parachlamydia acanthamoe- -a friendly to, referring to intracellular growth of Proto- bae and its life cycle within Acanthamoeba polyphaga: an electron chlamydia naegleriophila strain KNic within Naegleria micrograph study. Appl Environ Microbiol. 2002;68:3076–84. 9. Casson N, Entenza JM, Greub G. Serological cross-reactiv- amebae). The 16Sr RNA sequence (DQ635609) of KNic ity between different Chlamydia-like organisms. J Clin Microbiol. is 97.6% similar to that of P. amoebophila, making this or- 2007;45:234–6. ganism a member of the genus Protochlamydia. KNic does 10. Fang R, Raoult D. Antigenic classifi cation of Rickettsia felis by us- not grow on axenic media (1) but grows by 4 logarithms in ing monoclonal and polyclonal antibodies. Clin Diagn Lab Immu- nol. 2003;10:221–8. 60 h within A. castellanii. KNic exhibits a Chlamydia-like

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11. Thomas V, Casson N, Greub G. Criblamydia sequanensis, a new 15. Welti M, Jaton K, Altwegg M, Sahli R, Wenger A, Bille J. Devel- intracellular Chlamydiales isolated from Seine river water using opment of a multiplex real-time quantitative PCR assay to detect amoeba coculture. Environ Microbiol. 2006;8:2125–35. Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma 12. Kumar S, Tamura K, Nei M. MEGA3: Integrated software for mo- pneumoniae in respiratory tract secretions. Diagn Microbiol Infect lecular evolutionary genetics analysis and sequence alignment. Brief Dis. 2003;45:85–95. Bioinform. 2004;5:150–63. 13. Everett KD, Bush RM, Andersen AA. Emended description of the Address for correspondence: Gilbert Greub, Center for Research on order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Intracellular Bacteria, Institute of Microbiology, University Hospital Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new Center and University of Lausanne, 1011 Lausanne, Switzerland; email: genus and fi ve new species, and standards for the identifi cation of [email protected] organisms. Int J Syst Bacteriol. 1999;49:415–40. 14. Jaton K, Bille J, Greub G. A novel real-time PCR to detect Chlamyd- All material published in Emerging Infectious Diseases is in the ia trachomatis in fi rst-void urine or genital swabs. J Med Microbiol. public domain and may be used and reprinted without special 2006;55:1667–74. permission; proper citation, however, is required.

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